Natural Killer (NK) cells are large granular lymphocytes (LGL) that spontaneously lyse a limited range of target cells and that may be important in host defense against malignancy and viral infections. NK cells lack the T cell antigen receptor (Ti) and surface immunoglobulin (Ig). To examine transmembrane signalling events involved in NK cell activation and to identify the cell surface structures required for activation we have adapted for in vitro growth a rat LGL tumor, RNK-16, with features of NK cells. We have demonstrated that exposure of RNK-16 cells to susceptible targets generates inositol phosphates (InsPs) and a rise in intracellular free calcium ((Ca2+)i) in RNK-16. Monoclonal antibody (MAb) OX-34, which recognizes the rat homologue for human CD2, also stimulates these events, when OX-34 is cross- linked. Without cross-linking, OX-34 blocks the generation of InsP3 and cytotoxicity by RNK-16 cells. These studies provide the first demonstration of transmembrane signalling events associated with NK cell activation, and they implicate CD2 in the regulation of these signals. The objectives of our proposed studies are to define the biochemical events that lead to the activation of NK cells and to identify the cell surface structures that initiate activation. Our proposed studies will test three primary hypothesis. 1. The cell surface expression of CD2 is required for NK cell activity. These studies will examine a panel of tumor cells as well as purified LGL for the association of CD2 with NK activity and the inhibitory effect of anti-CD2. A major focus is the derivation of CD2- mutants of RNK-16, with collaborative efforts to reconstitute CD2 expression and killing by the mutants, by transfection with the CD2 gene. 2. Perturbation of CD2, alone or in combination with other signals, can activate NK cells, as assessed by degranulation. Degranulation of RNK-16 releases serine esterase. Experiments in 5 areas will examine the cell signals required for degranulation, focusing on perturbation of CD2 and the contribution of InsPs, (Ca2+)i, and the activation of protein kinase C. 3. NK cells express cell-surface molecules other than CD2 that can stimulate that generation of InsPs. CD2- mutants of RNK-16, and any other CD2- rat LGL tumors, will be examined for the generation of InsPs in response to lectins and to target cells. Mab will be developed against RNK-16 to test for stimulation of InsP3 through surface structure other than CD2.